Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility

The role of the pion cloud in the interpretation of the valence light-cone wavefunction of the nucleon

The pion cloud renormalises the light-cone wavefunction of the nucleon which is measured in hard, exclusive photon-nucleon reactions. We discuss the leading twist contributions to high-energy exclusive reactions taking into account both the pion cloud and perturbative QCD physics. The nucleon's electromagnetic form-factor at high $Q^2$ is proportional to the bare nucleon probability $Z$ and the cross-sections for hard (real at large angle or deeply virtual) Compton scattering are proportional to $Z^2$. Our present knowledge of the pion-nucleon system is consistent with $Z = 0.7 \pm 0.2$. If we apply just perturbative QCD to extract a light-cone wavefunction directly from these hard exclusive cross-sections, then the light-cone wavefunction that we extract measures the three valence quarks partially screened by the pion cloud of the nucleon. We discuss how this pion cloud renormalisation effect might be understood at the quark level in terms of the (in-)stability of the perturbative Dirac vacuum in low energy QCD.Comment: Expanded Discussion of Phenomenology and Spin Physic

The Intermediate Filament Network in Cultured Human Keratinocytes Is Remarkably Extensible and Resilient

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

The endothelial glycocalyx: composition, functions, and visualization

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging

The mammalian centrosome and its functional significance

Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome’s functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome’s role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome’s significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease

Costs of cancer care for use in economic evaluation: a UK analysis of patient-level routine health system data.

BACKGROUND: The rising financial burden of cancer on health-care systems worldwide has led to the increased demand for evidence-based research on which to base reimbursement decisions. Economic evaluations are an integral component of this necessary research. Ascertainment of reliable health-care cost and quality-of-life estimates to inform such studies has historically been challenging, but recent advances in informatics in the United Kingdom provide new opportunities. METHODS: The costs of hospital care for breast, colorectal and prostate cancer disease-free survivors were calculated over 15 months from initial diagnosis of cancer using routinely collected data within a UK National Health Service (NHS) Hospital Trust. Costs were linked at patient level to patient-reported outcomes and registry-derived sociodemographic factors. Predictors of cost and the relationship between costs and patient-reported utility were examined. RESULTS: The study population included 223 breast cancer patients, 145 colorectal and 104 prostate cancer patients. The mean 15-month cumulative health-care costs were £12 595 (95% CI £11 517-£13 722), £12 643 (£11 282-£14 102) and £3722 (£3263-£4208), per-patient respectively. The majority of costs occurred within the first 6 months from diagnosis. Clinical stage was the most important predictor of costs for all cancer types. EQ-5D score was predictive of costs in colorectal cancer but not in breast or prostate cancer. CONCLUSION: It is now possible to evaluate health-care cost using routine NHS data sets. Such methods can be utilised in future retrospective and prospective studies to efficiently collect economic data

Sertoli cells maintain leydig cell number and peritubular myoid cell activity in the adult mouse testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health